We are studying signaling networks in the retinal pigment epithelium (RPE) with special emphasis on lipid and retinoid metabolism pathways, differentiation/dedifferentiation pathways, and protection against oxidative or inflammatory stress. The role of signaling pathways in RPE differentiation and de-differentiation is an important focus of our research. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In addition, given the likely importance of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in the response of RPE cells to various signals, we are interested in determining changes in miRNA expression in RPE cells due to agents with which they are treated in our experiments. In the past year we have made progress in the following areas: 1) We are interested in elucidating how dysfunction of the RPE resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD), and to identify how inflammatory processes can be prevented. RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE ARPE-19 cell line under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-&#947;, IL-1&#946; and TNF-&#945; highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-&#954;B activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. These results indicate that resveratrol could potentially attenuate RPE inflammatory responses implicated in the pathogenesis of AMD. A manuscript describing these results was published in this reporting period. Current plans are to identify changes in gene regulation associated with these responses to inflammatory processes using microarray transcriptome analysis. 2) We continued a study to understand the mechanisms underlying dedifferentiation of RPE cells in primary culture and re-differentiation in the ARPE-19 RPE cell line. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research. On the one hand, using a cocktail of factors induced pluripotent stem (iPS) cells can be differentiated into cells sharing many aspects of RPE phenotype, and by rigorous culture methods, fetal RPE cells can be differentiated to retain or acquire aspects of native phenotype. On the other hand, explanted native RPE cells will lose important aspects of their RPE phenotype after a short time in culture. The various cell lines, such as the commonly used ARPE-19, do not have most native phenotypic features under common culture methods. What are the mechanisms regulating such gain or loss? Do mechanisms like epithelial-mesenchyme transition play a role in this process? We are particularly interested in the long-known but poorly understood loss by immortalized and primary RPE cells of expression of visual cycle enzymes. Understanding the mechanism underlying this down-regulation could be useful in ensuring that iPS-derived cells used for human transplant are fully competent to fulfill their intended role in restoring RPE function in treated eyes. Our experimental paradigm focuses i) on the loss of visual cycle phenotypic competence by adult bovine RPE cells explanted into primary culture, and ii) the regain of phenotypic competence by careful culture of relatively early passage ARPE-19 cells. Using these models we are analyzing expression of visual cycle and genes of other pathways and will correlate these to changes in gene regulation, RNA transcript expression and microRNA expression patterns. Striking changes in phenotype (cell morphology and melanization), gene expression, and biochemistry have been observed in ARPE-19 cells grown for 4 months or more, and indicate that ARPE-19 retains the plasticity to return to native-like RPE phenotype. We are comparing the results obtained from these experiments with ARPE-19 cell with those from parallel experiments with other RPE cell cultures, such as human RPE multipotent stem cells obtained from the Sally Temple group. The study is still ongoing and results from it were presented as a poster at a scientific meeting during the reporting period. 3) We began a project to examine the expression of secreted proteins (secretome) and exosomes in differentiated ARPE-19 cultured in DMEM with pyruvate for 4 months and exhibiting native-like RPE phenotype. We analyzed the secreted proteins from ARPE-19 cells grown in culture for 4 days or 4 months by MALDI TOF/TOF mass spectrometry and western blotting. Proteins known to be abundantly expressed in RPE were detected in cultured medium from 4-month cultures but were either absent or negligible from 4-day cultures. Cystatin C, among the most highly expressed proteins in native RPE, was abundantly detected in the medium from 4-month cultures, but absent in medium from 4-day cells. PEDF was also found to be abundantly secreted by 4-month cultures, but not by 4-day cultures. These results further establish that ARPE-19 can be differentiated to a native-like phenotype using optimized conditions. Results from this project were presented as a poster at a scientific meeting during the reporting period. We are also analyzing secretion from ARPE-19 cells via exosomes. We continue to collaborate within the LRCMB and with other laboratories and sections (Molecular Structure and Functional Genomics), as well as with extramural labs in the analysis of retinoid and other compounds. A manuscript on the latter was published during this reporting period.